There exists a need to make biological assays faster and simpler to perform with an overriding drive to make the processes cheaper yet maintain accuracy and reproducibility. This is due to a rapid increase in the number of research and diagnostic molecular probes available (e.g. new fluorescent reporter molecules) and the advantages in terms of information content of multiplexing such assays across a range of instruments. Increasingly there is a need for cell-, particle- and bead-based (and a combination of these units) assays in which the presence, for example, of cells with certain features indicates disease processes. Similarly, the demand and evolution of rational approaches in the search for bioactive molecules for new medicines has resulted in a need for low cost high-through-put screening (HTS) and the development of cell- and molecule-based assays, tools and arrays within the field of functional analysis.
Such assays require or are enhanced by the availability of methodologies for:    i) the manipulation of cells/particles, analytes and reagents in liquid and gel phases for processing purposes,    ii) the controlled delivery of fluorescent/bioluminescent molecules to cells/particles or the retention of the fluorescent/bioluminescent-associated properties of said particle/cells.    iii) the controllable immobilisation of said cells/particles for the purpose of analysis involving light collection    iv) the retention of cell viability and cell function for periods of time sufficient for the purposes of an analysis.
The present invention seeks to provide means for use in such methodologies.
A variety of hydrogels based upon thermoreversible polymers (in liquid form at elevated temperatures but in gel form at lower temperatures) are known, including natural gel-forming materials such as agarose, agar, furcellaran, beta-carrageenan, beta-1,3-glucans such as curdlan, gelatin, or polyoxyalkylene containing compounds.
The present invention exploits to the distinct advantages and properties of block polymer-based gels, such as polyoxypropylene-polyoxyethylene block polymer gels (PBP), which unusually undergo transition to liquid form upon temperature reduction. This property can be described as ‘reversed thermosetting’.
Selected properties of the PBP preparations, such as detergent properties and the mechanical and thermoreversible properties of hydrogels in general, have been documented and exploited in the art.
For example, reported uses and properties of PBP preparations include the following:
Surfactant Properties
Polyoxypropylene-polyoxyethylene block polymer (PBP) has been used as a non-ionic surfactant for detergents, dispersants, binders, stabilisers, defoaming agents, emulsifying agents to name but a few. At high aqueous PBP concentrations, beyond a transition temperature, a gel can form comprising amphiphilic block copolymer micelles. A commercial Pluronic® preparation F-127 (PEO99-PPO69-PEO99, with E and P being polyoxyethylene and polyoxypropylene, respectively) has been used in gel form for pharmaceutical preparations.
Reverse Agar and Biofilms
Gel-forming formulations of PBP have been described as ‘reverse agar’ in their use. The low temperature gelling formulation has been used to support the limited growth of micro-organisms in conventional microbiology applications. Such Pluronic®-based hydrogels have been used extensively to assay biocide treatments. Gel-trapped micro-organism populations mimic the localized high cell densities observed in biofilms and are subject to the similar nutrient and chemical gradients found within natural biofilms. Such prior art uses with respect to micro-organisms has revealed that PBP should have low toxicity in the stable gel form.
Molecular Sieve Properties
PBP gel form has the potential to form ordered micellar structures and has been used as a separation media for nucleic acids, indicating the coherent movement of molecules through the gel upon the passage of a continuous or pulsed electrical current. Microdevices have also been designed with sieving gels within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. Extensive comparisons have been made to compare different gel matrices on the basis of gel casting ease, reusability, and overall separation performance using for example a 100 base pair double-stranded DNA ladder as a standard sample.
Hydropads
Miniature size and high sensitivity of biochips is sought in diagnostics, testing, and research in medicine, veterinary science and applications, agriculture, toxicology, environmental monitoring, forensics etc. Three dimensional biochips comprising non-thermoreversible gels (hydropads) have been developed consisting of an array of three-dimensional gel elements on the hydrophobic surface of a microscope slide. For example, a gel-based biochip project was initiated in Engelhardt Institute of Molecular Biology of the Russian Academy of Science (EIMB) in 1989 resulting in the development of the ‘Immobilized Micro Array of Gel Elements’ on chip (or IMAGE chip), which can bear oligonucleotides, DNA, proteins, small compounds or cells fixed within semi spherical hydrogel pads. A simple two-step procedure has been developed for the large-scale manufacture of such chips. The gel pads can serve both as support for immobilisation and as individual nanoliter test tubes to carry out various specific interactions, chemical or enzymatic reactions. Chips have been produced that contain immobilized antibodies, antigens, enzymes, receptors, and different ligands.
Cell Encapsulation
Polymeric gels have also been explored as cell encapsulation materials for tissue engineering. Isolated mammalian cells and tissues have countless applications in medicine and biotechnology, yet protecting and nourishing cells either in vitro or in vivo while harvesting the desired products has proven difficult.
Drug and Dye Delivery
Previous clinical applications have revealed the use of hydrogels for the purpose of local delivery of pharmacologically active agents to tissues. In contrast, previous work on the “liquid less” cell staining by dye diffusion from gels (polyacrylamide or gelatin) has been restricted to the use of gel systems lacking the unique thermoreversible properties of PBP-based gels. Such studies have been described by Smolweski et al., 2001, Cytometry 44(4):355-60. Dye delivery to cells was observed but required a 2- to 4-fold increase in normal staining concentration of DNA dyes.